Predominant contribution of surface approximation to the mechanism of heparin acceleration of the antithrombin-thrombin reaction. Elucidation from salt concentration effects.
Predominant contribution of surface approximation to the mechanism of heparin acceleration of the antithrombin-thrombin reaction. Elucidation from salt concentration effects.
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DOI：
10.1016/s0021-9258(18)38125-0
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发表时间：
1991-04
期刊：
影响因子：
--
通讯作者：
S. Olson;I. Björk
中科院分区：
文献类型：
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作者：
S. Olson;I. Björk
肝素已被证明可通过促进蛋白酶和抑制剂在三元凝血酶-AT-肝素复合物中的初次相遇，加速抗凝血酶 III (AT) 对 α-凝血酶的灭活。本工作的目的是评估肝素诱导的 AT 构象变化以及凝血酶-肝素相互作用对肝素促进该三元复合物中凝血酶-AT 相互作用的相对贡献。这是通过比较在 pH 7.4、25 摄氏度和 0.1-0.35 M 氯化钠条件下三元复合物组装所涉及的二元和三元复合物相互作用中离子和非离子的贡献来实现的。作为盐浓度函数的二元复合物相互作用的平衡结合和动力学研究表明，凝血酶-肝素和 AT-肝素相互作用具有相似的大离子成分，但凝血酶-AT 相互作用主要是非离子成分。在肝素始终被 AT 饱和的条件下对三元复合物形成的停流动力学研究表明，在所有盐浓度下，三元复合物主要由游离凝血酶和 AT-肝素二元复合物组装而成。此外，凝血酶与与肝素结合的AT的三元复合物相互作用表现出与凝血酶-肝素二元复合物相互作用相似的大量离子成分。凝血酶二元和三元复合物相互作用的离子和非离子成分的比较表明：1）离子凝血酶-肝素和非离子凝血酶-AT二元复合物相互作用的加和贡献完全解释了凝血酶三元复合物相互作用的结合能，2）肝素诱导的AT构象变化对该结合能的贡献相对较小。因此，结果表明，肝素主要通过接近多糖表面上的蛋白酶和抑制剂来促进凝血酶和AT的相遇。进一步获得了凝血酶与 AT-肝素复合物结合的替代模式的证据，无论有或没有与 AT 复合的酶的活性位点。这一发现与凝血酶和 AT 的三元复合物相遇相一致，该三元复合物是由凝血酶与非特异性肝素位点结合介导的，然后沿着肝素表面扩散到与结合的抑制剂相邻的独特位点。
Heparin has been shown to accelerate the inactivation of alpha-thrombin by antithrombin III (AT) by promoting the initial encounter of proteinase and inhibitor in a ternary thrombin-AT-heparin complex. The aim of the present work was to evaluate the relative contributions of an AT conformational change induced by heparin and of a thrombin-heparin interaction to the promotion by heparin of the thrombin-AT interaction in this ternary complex. This was achieved by comparing the ionic and nonionic contributions to the binary and ternary complex interactions involved in ternary complex assembly at pH 7.4, 25 degrees C, and 0.1-0.35 M NaCl. Equilibrium binding and kinetic studies of the binary complex interactions as a function of salt concentration indicated a similar large ionic component for thrombin-heparin and AT-heparin interactions, but a predominantly nonionic contribution to the thrombin-AT interaction. Stopped-flow kinetic studies of ternary complex formation under conditions where heparin was always saturated with AT demonstrated that the ternary complex was assembled primarily from free thrombin and AT-heparin binary complex at all salt concentrations. Moreover, the ternary complex interaction of thrombin with AT bound to heparin exhibited a substantial ionic component similar to that of the thrombin-heparin binary complex interaction. Comparison of the ionic and nonionic components of thrombin binary and ternary complex interactions indicated that: 1) additive contributions of ionic thrombin-heparin and nonionic thrombin-AT binary complex interactions completely accounted for the binding energy of the thrombin ternary complex interaction, and 2) the heparin-induced AT conformational change made a relatively insignificant contribution to this binding energy. The results thus suggest that heparin promotes the encounter of thrombin and AT primarily by approximating the proteinase and inhibitor on the polysaccharide surface. Evidence was further obtained for alternative modes of thrombin binding to the AT-heparin complex, either with or without the active site of the enzyme complexed with AT. This finding is consistent with the ternary complex encounter of thrombin and AT being mediated by thrombin binding to nonspecific heparin sites, followed by diffusion along the heparin surface to a unique site adjacent to the bound inhibitor.